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Title: Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells

Abstract

7,8-Dihydro-8-oxo-2’-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [14C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 106 nucleotides, 5–6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis.

Authors:
 [1];  [1];  [1];  [2];  [1]
+ Show Author Affiliations
  1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Chemistry, Materials, Earth and Life Sciences Directorate
  2. Albert Einstein College of Medicine, Bronx, NY (United States). Dept. of Biochemistry
Publication Date:
Research Org.:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; National Institutes of Health (NIH); California Breast Cancer Research Program
OSTI Identifier:
1625418
Grant/Contract Number:  
AC52-07NA27344; RR13461; CA55861; 9KB-0179
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 36; Journal Issue: 1; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biochemistry & Molecular Biology

Citation Formats

Mundt, J. M., Hah, S. S., Sumbad, R. A., Schramm, V., and Henderson, P. T. Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells. United States: N. p., 2007. Web. doi:10.1093/nar/gkm1032.
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Mundt, J. M., Hah, S. S., Sumbad, R. A., Schramm, V., & Henderson, P. T. Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells. United States. https://doi.org/10.1093/nar/gkm1032
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Mundt, J. M., Hah, S. S., Sumbad, R. A., Schramm, V., and Henderson, P. T. Mon . "Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells". United States. https://doi.org/10.1093/nar/gkm1032. https://www.osti.gov/servlets/purl/1625418.
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@article{osti_1625418,
title = {Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells},
author = {Mundt, J. M. and Hah, S. S. and Sumbad, R. A. and Schramm, V. and Henderson, P. T.},
abstractNote = {7,8-Dihydro-8-oxo-2’-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [14C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 106 nucleotides, 5–6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis.},
doi = {10.1093/nar/gkm1032},
journal = {Nucleic Acids Research},
number = 1,
volume = 36,
place = {United States},
year = {Mon Nov 19 00:00:00 EST 2007},
month = {Mon Nov 19 00:00:00 EST 2007}
}
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https://doi.org/10.1093/nar/gkm1032
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